The six possible isomers of di-N-acetylchitotetraoses [AADD, ADDA, ADAD, DADA, DAAD, and DDAA, where D stands for 2-amino-2-deoxy-β-d-glucose (GlcN) and A for 2-acetamido-2-deoxy-β-d-glucose (GlcNAc)] were analyzed by ESI(+)-MSn. Collision induced dissociation via MSn experiments were performed for the sodiated molecules of m/z 769 [M+Na] + for each isomer, and fragments were generated mainly by glycosidic bond and cross-ring cleavages. Rules of fragmentation were then established. A reducing end D residue yields the O,2A4 cross-ring [M-59+Na]+ fragment of m/z 710 as the most abundant, whereas isomers containing a reducing end A prefer to lose water to form the [M-18+Na] + ion of m/z 751, as well as abundant O,2A4 cross-ring [M-101+Na]+ fragments of m/z 668 and B3 [M-221+Na]+ ions of m/z 548. MS3 of C- and Y-type ions shows analogous fragmentation behaviour that allows identification of the reducing end next-neighbour residue. 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